Transformation of plant cells by an Agrobacterium mediated method involves exposing plant cells and tissues to a suspension of Agrobacterium cells that contain certain DNA plasmids. These DNA plasmids have been specifically constructed to contain transgenes that will express in plant cells (U.S. Pat. No. 5,034,322). Most often, one or more of the transgenes is a positive selectable marker transgene that permits plant cells to grow in the presence of a positive selection compound, for example an antibiotic or herbicide. These cells can be further manipulated to regenerate into whole fertile plants.
The methods for introducing transgenes in plants by an Agrobacterium mediated transformation method utilizes a T-DNA (transfer DNA) that incorporates the genetic elements of a transgene and transfers those genetic elements into the genome of a plant. The transgene(s) are constructed in a DNA plasmid vector and are usually bordered by an Agrobacterium Ti plasmid right border DNA region (RB) and a left border DNA region (LB). During the process of Agrobacterium mediated transformation the DNA plasmid is nicked by VirD2 endonuclease at the right and left border regions and the T-DNA region is inserted into the plant genome. The integration of the T-DNA into the plant genome generally begins at the RB and continues to the end of the T-DNA, at the LB. However, the endonucleases sometimes do not nick equally at both borders. When this happens, the T-DNA that is inserted into the plant genome often contains some or all of the plasmid vector DNA. This phenomenon is referred to as border read-through. It is usually preferred that only the transgene(s) located between the right and left border regions (T-DNA) is transferred into the plant genome without any of the adjacent plasmid vector DNA (vector backbone). The vector backbone DNA contains various plasmid maintenance genetic elements, e.g., origin of replications, bacterial selectable marker genes, and other DNA fragments not desirable in commercial crop products for regulatory issues.
Considerable resources are directed at screening the genome of transgenic crop plants for the presence of the vector-backbone DNA. Methods such as polymerase chain reaction (PCR) and Southern blot analysis are most often employed to identify the extraneous vector backbone DNA. These methods are time consuming and expensive for large scale screening work. Vector backbone DNA can be incorporated by read through of the left border region or may integrate into the plant genome independently of the T-DNA (Kononov, et al., Plant J. 11, 945-957, 1997). The transgenic plants that are found to contain the vector backbone DNA are generally not viable for commercialization. Substantial efforts are wasted regenerating plants from plant cell culture that have no commercial potential. It would be useful to have a DNA construct and a method that would greatly reduce the occurrence of vector backbone DNA in the genome of transgenic plants. Fewer transgenic plants would have to be produced if a greater number were free of vector backbone DNA. Hence, fewer assays would have to be performed to confirm that the backbone DNA is absent.
Hanson, et al. (U.S. Pat. No. 6,521,458) describes a DNA construct that contains a lethal gene in the vector backbone that, when expressed, kills the plant cell. However, the control of the expression of lethal gene products in bacteria and plant cells can be problematic. Lethal gene expression must be controlled by various genetic elements to prevent expression in bacteria and in non-target plant cells and tissues. The use of non-lethal negative selectable marker genes for plant cells in the backbone would be a substantial improvement over the use of lethal genes. Non-lethal negative selectable marker genes can provide a visual means to distinguish plant cells and tissues that are expressing the non-lethal negative selectable marker gene products, the selection of the plant cells and plants is more controllable, and plant cells containing the non-lethal negative selectable marker genes are potentially rescuable. The gene used for this purpose can be any gene affecting plant cell division, shoot elongation or producing pleiotropic shoot or leaf phenotypes.
Scorable maker genes for example beta-glucuronidase (GUS) (Kononov, et al., Plant J. 11, 945-957, 1997), can provide a means to detect the presence of backbone DNA, but do not provide a means to select against the cells that contain them and the assay is tissue destructive. Negative selectable marker genes that are conditional lethal can also be used in the backbone DNA. Representative examples of other conditional lethal gene products include: E. coli guanine phosphoribosyl transferase that converts thioxanthine into toxic thioxanthine monophosphate (Besnard et al., Mol. Cell. Biol. 7:4139-4141, 1987); alkaline phosphatase, which will convert inactive phosphorylated compounds such as mitomycin phosphate and doxorubicin-phosphate to toxic dephosphorylated compounds; fungal (e.g. Fusarium oxysporum) or bacterial cytosine deaminase (codA) that will convert 5-fluorocytosine to the toxic compound 5-fluorouracil (Mullen, PNAS 89:33, 1992); carboxypeptidase G2 which will cleave the glutamic acid from para-N-bis (2-chloroethyl) aminobenzoyl glutamic acid, thereby creating a toxic benzoic acid mustard; and Penicillin-V amidase, which will convert phenoxyacetabide derivatives of doxorubicin and melphalan to toxic compounds (see generally, Vrudhula et al., J. of Med. Chem. 36(7):919-923, 1993; Kem et al., Canc. Immun. Immunother. 31(4):202-206, 1990); and phosphonate monoester hydrolase, pehA (U.S. Pat. No. 5,254,801). However, exogenous substrates must be added in order to provide the toxic product that is lethal to the cell containing the backbone DNA. The present invention does not require adding additional substrates to the culture media or exogenously treating the plant culture cells with a substrate as needed for the conditional lethal gene product.
Plant hormone signal transduction genes and hormone biosynthetic pathway genes can be used as selectable marker genes for plant transformation and in a method to produce marker free transgenic plants (U.S. Pat. No. 6,326,192). However, these genes must be removed if the plants are to be further developed as commercially viable plants as described. The genes and compositions illustrated therein can be used in the present invention as non-lethal negative selectable marker genes of the vector backbone DNA.
Gene products that metabolize endogenous plant cell substrates can function as metabolic interference gene of the present invention. For example, a sacB gene, encoding levansucrase and responsible for neutral polyfructan (levan) synthesis using sucrose as a substrate, was identified in many bacteria such as Bacillus spp., Erwinia spp. etc. Transgenic plants expressing sacB gene aimed at increasing drought resistance or sink strength were previously reported in tobacco, potato, sugar beet, maize and ryegrass (Ebskamp et al. Bio/Technol. 12, 272-275,1994; van der Meer at al. Plant Cell, 6, 561-570, 1994; Caimi et al. Plant Physiol. 110, 355-363,1996; Rober et al. Planta, 199, 528-536,1996; Ye et al. Plant Cell Rep., 20: 205-212, 2001). However, when the vacuole targeted sacB gene driven by CaMV 35S promoter was repeatedly transformed into tobacco and ryegrass, only stunted plants were recovered (Ye et al. 2001). In corn, the sacB expressing kernels disturbed grain filling and resulted in shrunken seeds with very low germination frequency (Caimi et al. 1996). In potato, the expression of the sacB gene in tubers lead to smaller tubers (Rober et al. 1996). These results revealed that expression of the sacB gene severely inhibit plant cell and tissue development.
Other genes encoding metabolic interference enzymes, such as yeast invertase, yeast trehalose-6-phosphate synthase may also be used in same way. It was reported that expression of yeast invertase (Suc2, Carlson et al., Nucleic Acids Res. 11 (6), 1943-1954, 1983) in tobacco and Arabidopsis strongly inhibit shoot elongation and root development (Sonnewald et al. Plant J. 1:95-106, 1991), and constitutive expression of yeast trehalose-6-phosphate synthase (TPS1, Bell et al. Eur. J. Biochem. 209 (3), 951-959 (1992) in tobacco exhibited stunted growth and lancet-shape leaves (Romero et al. Planta 201:293-297, 1997). The metabolic interference genes, for example, a polynucleotide encoding a levansucrase, an invertase or a trehalose-6-phosphate synthase are useful as non-lethal negative selectable marker transgenes in the present invention.
The present invention has incorporated a non-lethal negative selectable marker transgene into the vector backbone DNA of a DNA plasmid used to transform plant cells. These transgenes are designed to express a non-lethal gene product in plant cells that contain the vector backbone DNA of the DNA plasmid. The gene products of the non-lethal negative selectable marker transgene are involved in plant hormone biosynthesis pathways, plant hormone substrate diversion, plant hormone degradation, or metabolic interference. The use of these DNA plasmids to transform plant cells provides for enhanced production of commercially viable plants.